Expedited Delivery

Transfecting mammalian cells with a recombinant protein expression vector fulfills many research needs, but there are applications that require direct intracellular delivery of proteins. Brute force (electroporation or microinjection) can do the trick; alternatively, there are a number of lipid-based formulations for protein delivery. However, one of the most popular approaches relies on the use of protein transduction domains: basic peptide sequences found naturally within proteins such as HIV-1 Tat and Drosophila Antennapedia. Although the exact mechanism by which these sequences work has been debated, published reports indicate that adding hydrophobic, negatively charged counterions can enhance membrane translocation of these arginine-rich peptides—but most of these data derive from tests with an artificial membrane system. In a direct analysis of this phenomenon by microscopic monitoring of living cells, Takeuchi et al., convincingly show that pyrenebutyrate, one such counterion, can substantially increase the efficiency of cytosolic delivery. Significantly, the presence of pyrenebutyrate appeared to redirect a fluorophore-octaarginine peptide from an endosomal pathway to a diffuse cytosolic signal suggestive of direct membrane translocation. This increase in transduction efficiency was possible without evident damage to the plasma membrane or measurable cytotoxicity (pyrenebutyrate is freely diffusible and can be washed out of the cells postdelivery). Furthermore, the pyrenebutyrate enhancement effect was also observed with octaarginine-EGFP and permitted efficient protein transduction of primary neuronal cells. Takeuchi et al., also confirmed that the delivery strategy does not interfere with the biological activity of an octaarginine-conjugated cargo, as demonstrated by observing that cells behaved as expected when transduced with PAD, a proapoptotic domain peptide. It is important to note that the method must be performed in serum-free media; however, given that only a few minutes are required for efficient transduction, cells can be quickly returned to their favored incubation medium. In addition to its speed and convenience, the strategy appears to be broadly applicable (HeLa, COS, and RAW264.7 were among the cell lines tested) and should prove particularly useful for delivering chemically modified proteins, which could not otherwise be expressed intracellularly. –ND

-Takeuchi et al. 2006. Direct and rapid cytosolic delivery using cell-penetrating peptides mediated by pyrenebutyrate. ACS Chemical Biology 10.1021/cb600127m.

Focused on Peptides

The discovery of the power of mass spectrometry for proteomics research has enabled the extremely rapid analysis of many more proteins than was previously possible, but these advances have not come without their problems. The innate complexity of the samples analyzed—often either cell lysates or tissue extracts—has strained the limits of the equipment, frustrating attempts to obtain greater resolution. Popular and simple preseparation techniques such as two-dimensional polyacrylamide gel electrophoresis (PAGE) can reduce this complexity through isolation and testing of only the protein fragments of interest. As a potential alternative to PAGE techniques, a group in the Netherlands proposes using isoelectric focusing (IEF) of peptides prior to mass spectrometry as a means to separate peptides and gain valuable information regarding their isoelectric points (pI). The pI measurement for a specific peptide has been suggested as a useful datum that can validate a peptide's identity. This technique of “data filtering” was tested using Drosophila nuclear extracts separated on immobilized pH gradient (IPG) IEF strips and examined by nanoflow liquid chromatography coupled to linear ion trap or Fourier Transform (FT) mass spectrometry. Results were compared with separation by standard gel electrophoresis methods, namely SDS-PAGE, and the same mass spectrometry analysis. The comparison showed clearly that the IEF procedure performed well, significantly increasing the number of peptides confidently identified over SDS-PAGE, while maintaining a low false positive rate. This highlights the superior resolving power of IEF, which provides an excellent means to prescreen peptides and obtain high quality data equivalent to that achievable with higher end FT devices, even while using comparatively low end machines such as linear ion trap mass spectrometers. -SS

-Krijgsveld et al. 2006. In-gel isoelectric focusing of peptides as a tool for improved protein identification. Journal of Proteome Research [Epub ahead of print, June 9, 2006].

ELP Is on the Way

Working with Affymetrix GeneChips® in Arabidopsis, West et al., demonstrate an efficient new approach for haplotyping recombinant inbred lines (RILs). The strategy relies upon two different sets of markers. The first marker type derives from the treatment of gene expression levels as quantitative traits. These expression level polymorphisms (ELPs) are determined by so-called eQTLs and are defined for mapping purposes as GEMs, or gene expression markers. The second set of markers comprises sequence polymorphisms (called single feature polymorphisms or SFPs in Arabidopsis). Both sets of markers can be queried using the short oligonucleotide probes of GeneChips. Ordinarily, GEMs would be assayed using RNA, and SFPs would be detected by using genomic DNA as input. Intent on saving time and expense, West et al., show how both sets of markers can be obtained from RNA-based GeneChip hybridizations alone, without the need for separate genotyping. GEMs rely upon parental line differences in signal in each member of the gene's specific probe set; the authors describe the filtering process necessary to derive meaningful markers for screening the progeny. Use of GEMs improved marker density from 1 per 10.8 cM (from an original set of 38 microsatellite markers) to 1 per 1.7 cM. SFPs were identified by looking for differences in the hybridization values for single oligonucleotide members within a gene probe set, meaning that SFPs remain independent of a gene's expression level. In combination with the existing microsatellite markers, the SFPs permit the construction of the genetic map with an average resolution of 1 marker per 0.64 cM. West et al., are employing their haplotype data and the corresponding genetic map to decipher the eQTLs underlying ELPs in Arabidopsis; however, the strategy should also be applicable to hasten eQTL analysis in other organisms. –ND

-West et al. 2006. High-density haplotyping with microarray-based expression in single feature polymorphism markers in Arabidopsis. Genome Research 10.1101/gr.5011206.

An Array of Possibilities

As all roads lead to Rome, so all—or at least, most—cell signals lead to the nucleus. There, transcription factors are activated to respond to external information by up- or down-regulating gene expression through direct binding to specific, and frequently highly conserved, DNA sequence motifs. Now that the genomes for many organisms are known, and their proteomes under increasing scrutiny, the push to match all transcription factors with their corresponding recognition sites is intensifying. Numerous powerful, high-throughput techniques have been developed to determine the DNA sequence to which known, or suspected, transcription factors bind. These include SELEX, as well as a whole family of methods based on the ChIP assay. Rare, however, are such techniques that do the opposite, namely identify the transcription factors that bind to suspected regulatory motifs. Now, Michael Snyder and his group at Yale, in collaboration with Mark Johnston's lab at Washington University, have come up with an array solution that does just this. They arrayed 282 yeast proteins that are known or suspected to have DNA binding capacity and interrogated them with probes covering 75 different regulatory motifs found to be evolutionarily conserved. Proteins known to be nonspecific DNA binders were actively excluded from the array. Hybridization with control probes in which the conserved sequences had been mutated was performed to identify and exclude additional nonspecific binding activity. Even though the assay is currently limited in its ability to truly replicate in vivo, or even cell culture, conditions (such as oligomerization of transcription factors or the presence of essential cofactors), it still enabled the identification of a number of DNA-protein interactions not previously described and which could be confirmed by more biologically relevant experiments. With the potential to apply to any organism, this technology provides a promising, high-throughput means to identify and more fully characterize the multitude of DNA-protein interactions involved in all facets of cell function regulation. –SS

-Ho et al. 2006. Linking DNA-binding proteins to their recognition sequences by using protein microarrays. Proceedings of the National Academy of Sciences of the USA 103(26):9940-9945.